Conventional two‐dimensional as well as recently established three‐dimensional cell culture systems exhibit a high modulus of elasticity, yet, without representing the stiffness of human organs. Previously, we established a protocol for the culture of primary rat hepatocytes on a silicone of low stiffness, which sustained their physiological functions. This tool may readily serve as validation system for pharmacological and toxicological studies of agents and compounds. In terms of the development of human‐based tests to evaluate the potential risk of novel drugs, the method was adjusted for use of human adipose tissue‐derived mesenchymal stromal cells (hAT‐MSC).Aim of the study was to optimize culture conditions for hAT‐MSC on fibronectin‐coated silicone layers with various elastic moduli (50 – 500 kPA) mimicking the stiffness of human organs including different surface modifications to increase hydrophilic properties. The metabolic activity (MTT assay) and growth parameters were analysed and compared to the conventional cell culture on polystyrene (E‐modulus 103 MPa).hAT‐MSC were cultured for 10 days on five different fibronectin‐coated medically approved silicone layers. The impact of surface modifications of the silicone using sol‐gel‐technique, fluorination and plasma treatment on the cells was determined by the development of cell counts, viability and metabolic activity. hAT‐MSC attached to all 5 different silicone layers and formed cell layers after 10 days. Neither the silicones with surface modifications nor the untreated silicone displayed significant differences in cytotoxicity: the percentage of dead cells in cultures were 7% ± 2.3% vs. 7.2% ± 1%. We identified one culture displaying significantly higher cell counts as compared to the standard culture: based on identical initial cell counts, the growth rate was about 2.5‐fold higher on plasma‐treated silicone with an E‐modulus of 50 kPa. Cells cultured on plasma‐treated silicones with E‐moduli of 50 kPa or 500 kPa showed also significantly higher amounts of metabolic activity as compared to the classical culture: 2.76‐fold (50 kPA) and 2.83‐fold (500 kPa).With these results, two medically approved silicones treated with plasma were identified for improving culture of hAT‐MSC. Thus, the silicone layers represent an important tool for the development of cell cultures featuring organ‐like stiffness and improving physiological functions.Support or Funding InformationThe study was supported by the European Regional Development Fund and the Free State of Saxony (SAB) 100282466 to J.H. and P.S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.